禽新型黄病毒RTLAMP检测方法的建立

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (4): 659-663.

禽新型黄病毒RTLAMP检测方法的建立

李兆龙1,2,陈仕龙1,2,林锋强1,2,王劭1,2,程晓霞1,2,朱小丽1,2,陈少莺1,2*

1.福建省农业科学院畜牧兽医研究所，福州 350013； 2. 福建省畜禽疫病防治工程技术研究中心，福州 350013

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-04-25 发布日期:2012-04-25
通讯作者: 陈少莺

Establishment of Rapid Reverse Transcription Loop Mediated Isothermal Amplification for Detection of Avian Novel Flavivirus

LI Zhaolong1,2, CHEN Shilong1,2, LIN Fengqiang1,2, WANG Shao1,2,
CHENG Xiaoxia1,2, ZHU Xiaoli1,2, CHEN Shaoying1,2*

1. Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2. Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China

Received:1900-01-01 Revised:1900-01-01 Online:2012-04-25 Published:2012-04-25
Contact: CHEN Shaoying

摘要/Abstract

摘要： 根据环介导等温扩增技术(LAMP)，建立了一种适用于禽新型黄病毒的逆转录环介导等温扩增(RTLAMP)快速检测方法。根据禽新型黄病毒E蛋白基因序列，在保守区设计了一套针对禽新型黄病毒基因8个区域的6条特异性引物，并对反应条件进行优化。结果表明该方法对NDRV、GPV、MDRV、NDV、ILTV、FPV均无扩增反应，并可通过反应液是否有沉淀或向反应液中加入荧光染料来对结果进行可视化观察; 扩增反应只需要在常规水浴锅中进行，45 min内可完成反应; 该方法对禽新型黄病毒RNA的最小检测限为1 pg，灵敏度是一步法RTPCR方法的100倍。本研究建立的RTLAMP方法简便、快速、灵敏、特异，适合在基层进行禽新型黄病毒的快速检测。

关键词: 禽新型黄病毒, RTLAMP, 检测方法

Abstract: A reverse transcription loop mediated isothermal amplification (RTLAMP) assay was developed for detection of avian novel flavivirus virus. According to the sequences of avian novel flavivirus virus E protein gene, six primers specific to the eight sites of novel flavivirus virus gene were designed, and the reaction conditions were optimized. The results showed that the assay had no cross reaction with NDRV, GPV, MDRV, NDV, ILTV and FPV. The assay was performed in water bath within 45 minutes and the amplification result was visualized by adding SYBR Green Ⅰ or inspecting the white sediment. The detection limit of RTLAMP assay was 1 pg, which was 100 fold higher than that of one step RTPCR. The detection in clinical samples was 100% compliance with RTPCR. These results suggested that the newly developed RTLAMP assay is a simple and specific method for rapid detection of avian novel flavivirus virus in field conditions.

Key words: avian novel flavivirus, loopmediated isothermal amplification, diagnosis method

李兆龙;陈仕龙;林锋强;王劭;程晓霞;朱小丽;陈少莺;. 禽新型黄病毒RTLAMP检测方法的建立[J]. 畜牧兽医学报, 2012, 43(4): 659-663.

LI Zhaolong;CHEN Shilong;LIN Fengqiang;WANG Shao;CHENG Xiaoxia;ZHU Xiaoli;CHEN Shaoying;. Establishment of Rapid Reverse Transcription Loop Mediated Isothermal Amplification for Detection of Avian Novel Flavivirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(4): 659-663.

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